Abstract The proposed research project focuses on the thrombomodulin-dependent activation of protein C by thrombin as a key regulatory feedback loop of the coagulation response. Our interest in this reaction stems from its physiological relevance, the lack of a molecular understanding of its mechanism and the translational opportunities that might ensue from advances in basic knowledge. Investigation of protein C is motivated by our recent success in the crystallization of prothrombin and characterization of its structure in solution, as well as by new reagents developed in the lab, i.e., a derivative of protein C devoid of the auxiliary Gla and EGF domains (miniPC) expressed in E. coli for isotope labeling and a fusion protein (FP) of thrombin with the EGF456 domains of thrombomodulin that recapitulates the structural and functional properties of the thrombin- thrombomodulin complex. Our guiding hypothesis is that thrombomodulin (the cofactor) functions by optimizing the environment of the catalytic Ser of thrombin (the enzyme) and by exposing the Arg residue at the site of activation of protein C (the substrate). Studies under specific aim 1 will pursue X-ray, single molecule Frster resonance energy transfer and small angle X-ray spectroscopy of protein C free and bound to FP. Additional details on the conformation of protein C and of its activation domain in solution will be obtained by NMR measurements of miniPC. Success of these studies will provide unprecedented and much needed structural information on protein C and will significantly expand our understanding of the role of conformational plasticity in the mechanism of zymogen activation in this and other members of the trypsin family. Structural studies will be complemented by mutagenesis studies under specific aim 2. The effect of thrombomodulin on the catalytic Ser of thrombin will be investigated either directly through Thr, Cys and Tyr substitutions, or indirectly by removal of potential steric hindrance in the active site region. The effect of thrombomodulin on the site of cleavage of the activation domain of protein C will be investigated with substitutions that disengage the side chain of R169 from neighbor interactions through perturbation of backbone and side chains. Success of our studies will advance our basic knowledge on a key regulatory reaction of the coagulation cascade and will offer a relevant template for the analysis of other cofactor-assisted interactions in the blood coagulation, complement and fibrinolytic cascades.